2.1. Lipid Phase Behaviour and Aqueous Lipid Particle Dispersions
Lipids of vegetable origin consist of polar and non-polar components. The non-polar lipids are of great importance in foods, providing raw materials for fats or oils. The polar lipids are mainly used in order to achieve special functional properties, such as emulsification. The term polar lipid is used in this context based on the interaction with water; thus lipids forming aqueous phases are termed polar, whereas a lipid sample which do not form aqueous phases are termed non-polar. Polar lipids of vegetable origin are obtained at the so-called degumming step in the refining process of oil crops, such as soy bean oil. These lipids are dominated by phospholipids. Another source of polar lipids is cereals, containing a mixture of phospholipids and galactolipids. In this invention we have started from ethanol-water solutions of mixtures of galactolipids and phospholipids, with focus on ethanol extracts of oats, in which the polar lipids have been enriched by fractionation. At a certain concentration of the lipids in ethanol-water mixtures of a particular composition, a fluid was formed containing aggregates of a liquid-crystalline state which as far as we know never earlier has been reported in lipids. The molecular organization in this fluid and transformations at water dilution/ethanol evaporation is utilized in our invention.
Dispersions of the lamellar liquid-crystalline phase in an aqueous environment, which were termed liposomes, were first described in the 1960s. These particles consist of spherically concentric lipid bilayers alternating with water layers. They are formed from the lamellar liquid crystalline-phase, the L-alpha phase, by mechanical dispersion in excess of water. Thus a prerequisite is that the L-alpha phase can coexist in equilibrium with a water phase. The liposomes prepared from the L-alpha phase consist of several concentric bilayers, and are also termed multilamellar vesicles. There are also specific methods described in the literature for preparations resulting in unilamellar vesicles, which have been applied particularly in drug delivery. This invention describes a new method to prepare monodisperse aqueous dispersions of vesicles of galactolipids and phospholipids of very small size, and the use of such particles.
There is an extensive literature on manufacture and use of liposomes. The dominating lipids used are phospholipids and the most common applications involve delivery of biologically active components for pharmaceutical and food use.
Liposomes are usually formed by mechanical homogenization of the lamellar liquid-crystalline phase in excess of water, including ultrasonification and use of valve homogenizers. Commonly used reported methods in the literature involve the dry film method and the ethanol injection method. In order to obtain liposomes or vesicles of uniform size, molecular sieve methods have been used.
There are also other liquid-crystalline phases in lipid-water systems. The most important ones are beside the earlier mentioned L-alpha phase, the inverse hexagonal liquid-crystalline phase, also termed HII-phase, and cubic phases. These can also form aqueous dispersions. The introduction of the cryo-TEM method has allowed a detailed structural description of both inverse cubic and hexagonal particles in water (Langmuir 12 (1996) 4611-4613 and 13 (1997) 6964-6971. J. Gustaysson, H. Ljusberg-Wahren, M. Almgren, K. Larsson respectively). In the text below the lamellar liquid-crystalline phases is termed L-alpha phase, and the inverse hexagonal liquid-crystalline phase is termed HII-phase.
During the development of a preparation process of vesicle dispersions of polar lipids prepared from oats we realized that the same process could be applied in order to prepare uniform particle dispersions of oil-in-water and furthermore by introducing monoglycerides into mixtures of phospholipids and galactolipids uniform particle dispersions of cubic-phases could also be efficiently prepared.
We have developed a step-wise ethanol/water dilution process in order to prepare colloidal dispersions of polar lipids and it is described in detail here in the case of oat lipid fractions. This process was introduced mainly to avoid formation of the HII-phase at direct water exposure. This method was found to produce unilamellar vesicles in a smaller size range than earlier described. Prior art process required extremely pure polar lipid fractions as starting material. Our new process enables use of less purified staring material and at the same time accepts a higher load of active ingredient in delivery systems, while maintaining or improving the functional properties of the dispersion.
2.1.1 Prior Art and their Relations to Our Invention
As we use ethanol solutions we will consider here prior art for lipid particle preparation involving ethanol. A method based on ethanol solution droplets exposed to a water phase that has become important for liposome preparation was introduced long time ago (S. Batzri and E. Korn: Biochim. Biophys. Acta 298 (1973) 1015-1019). They injected an ethanol solution of phospholipids into the water phase. Large-scale production using this method has also been reported, cf. (R. Naeff: Adv. Drug Delivery Rev. 18 (1996) 343-347), including preparation of tuneable particle size distributions controlled by needle diameter in combination with hydrodynamic pressure (P. Prahan, J. Guan, P. G. Wang, L. J. Lee, R. J. Lee: Anticancer Res. 28 (2008) 943-947).
The use of ethanol for preparation of cubic particles was introduced by Spicer and co-workers and is further discussed below in connection with such particles.
These applications of direct water-dilution of ethanol solutions of the lipids are fundamentally different from our ethanol-water dilution process to produce particle dispersions. The earlier described prior art methods cannot be applied to our galactolipid and phospholipid systems as gel phases with the hexagonal HII-phase will be formed irreversibly.
In our case the ethanol-water dilution of the ethanol-lipid mixture must be carefully controlled so as to avoid formation of the hexagonal HII-phase. At ethanol concentrations higher than a critical ethanol concentration, the HII-phase cannot form. By dilution of an oil containing polar lipids, using an ethanol solution with a concentration close to the critical concentration, it is possible to separate the lamellar phase directly without formation of any HII-phase.
The prior art processes require extremely pure polar lipid fractions as starting material. Our new process enables use of less purified staring material and at the same time the particles can carry a higher load of active ingredients in delivery applications, while the functional properties of the dispersion are maintained or improved.
The interaction of cereal lipids with water has been analysed in detail in the case of wheat lipids, which resulted in the ternary phase diagram shown in FIG. 1. Similar phase equilibria occur also for oat lipids as shown below. The phase diagram shows phase equilibria of wheat lipids and water at room temperature. The polar and non-polar wheat lipid components were first separated and then reconstituted in different proportions before equilibration with water. Two liquid-crystalline phases exist—the lamellar (LC-L) and the inverse hexagonal (LC-H) and one liquid phase (L) as identified by X-ray diffraction. The broken line shows the composition polar/non-polar lipids in the wheat endosperm (after Larsson et al. 2006 Lipids: Structure, Physical properties and Functionality, The Oily Press)
The phase equilibria and physical structures of lipid extracts from oats are in general agreement with the wheat lipid system (G. Jayasingha, K. Larsson, Y. Miezis and B. Sivik. J. Dispersion Sci. Techn. 12 (1991) 443).
From FIG. 1, it can be seen that at a polar lipid concentration above 65 lipid % and addition of water the hexagonal phase HII is formed. The hexagonal phase creates a gel and this gel is extremely viscous and impossible to disperse into small particles.
However, the starting material in this invention contains ethanol. Thus a fourth dimension is introduced. In oat lipids with ethanol the formation of HII-gel occurs when the lipids are diluted in water, see Example 1. Once formed it takes very long time to dissolve the HII-gel. At room temperature the time scale is years.
At direct dilution of the oat lipid fractions of this invention, the HII-phase is always formed before the L-alpha phase, see Example 1, and therefore the prior art methods to produce liposomes in phospholipid systems cannot be applied. When the HII-phase is formed in phase diagrams of phospholipids, there appears to be an existence region of the L-alpha phase at lower temperature, where water dilution towards liposomes can take place.
By diluting the lipids with an ethanol-water solution according to a carefully designed protocol, defined in this invention, formation of the HII-particles can be avoided. This procedure is a prerequisite for the formation of the very small particles presented in this invention. In this way we can produce very small particles using lipid-mixtures of wide composition variations and with high concentrations, and this allows us to load more active components in the particles compared to prior art. The lipids used here form the liquid crystalline phase at and above room temperature so processing can be done at room temperature. The carefully designed ethanol-water dilution protocol also enables production of polar lipid fractions free from ethanol in a reproducible way without HII-particles. These polar lipid fractions have a very high and stable capacity to make oil-in-water emulsions.
There are only a few reported applications of cereal lipids and galactolipids which are discussed in detail below:    1. WO9520944 (U.S. Pat. No. 6,022,561) describes “bilayer preparations” which are prepared from chromatographically purified cereal extracts resulting in a polar lipid concentration of 100% and a concentration of DGDG above 67% (see their table 1) for producing carrier particles for active ingredients, in the form of either a gel or liposomes which are shown to be multilamellar. These very pure polar lipids could be mixed with water without formation of HII. After ultrasonication the average particle size of the vesicles was 144 nm.    The small unilamellar vesicles we obtain have average particle size below 100 nm. In addition the cubosomes we describe cannot be formed by their technology. Note also that we achieve much smaller particles using polar lipids with a much lower purity.    2. WO 2009/131436A1 describes a yoghurt product with an oil-in-water emulsion for satiety enhancement, and in the formulation of the complex mixture they use galactolipids as emulsifier (claim 3), and also oat lipids are mentioned (claim 4). The particles size for the oil-in-water (O/W) stabilized with an additional stabilizer, was such that at least 75% of the particles should be smaller than 1000 nm. Thus, their process will never obtain the O/W emulsions we describe.    3. WO 2009/068651 A1 describes an O/W emulsion for lowering cholesterol by phytosterols as a component in all claims. Galactolipids are used as emulsifier. Particle sizes are not given.    4. Li X. et al, Preparation and stability of DGDG as emulsifier for sug-microemulsions, Zhongguo zhongyao zazhi 2009, 34(17) 1272-1276 (AN2009:1571859) describes that digalactosyl diglycerides are used to make a submicro-emulsion of “bay oil” as a model drug. The particle size after high pressure homogenisation was 168 nm. Thus the particles are significant larger than the particles from our process.2.1.2 Conclusion on Lipid Particles
Within a wide range of proportions between galactolipids and other components of oat or other cereal lipids, the formation of an HII-phase is a serious complication for formation of uniform vesicle dispersions. This invention provides a solution to this problem.
During the development of the preparation process focused towards vesicle dispersions in water of oat lipids it was realized that this process also can be applied to preparation of polar lipid fractions free from ethanol and HII crystals, oil-in-water emulsions and furthermore cubic liquid-crystalline particles in water can be prepared when monoglycerides are introduced into the cereal lipid mixture.
2.2 Appetite Regulation
Obesity is a big and increasing problem in the western society. It has been estimated that $45 billion of US healthcare costs, or 8% per annum of total healthcare spend, is a direct result of obesity. Traditional approaches to long term weight management such as diet and exercise have proved ineffective alone to control the spread of obesity. Today, more than ever, there is a considerable interest in developing safe, effective methods for treatment of obesity.
Pharmacological approaches to the treatment of obesity have focused on either developing drugs that increase energy expenditure or drugs that reduce energy intake. One approach to the reduction of energy intake is to reduce the body's ability to digest and adsorb food, in particular fat. The key enzyme involved in digestion of fat is pancreatic lipase that is secreted by pancreas into the gut lumen.
The lipase inhibitor lipstatin has formed the basis of the anti-obesity drug, orlistat. Orlistat and its use in inhibiting pancreatic lipase and treating hyperlipaemia and obesity is disclosed in EP129748 (Hoffmann-la Roche, 1984). The use of orlistat (Xenical) as a drug against obesity is well established (Sjöström L. et al., Lancet 352: 167-172, 1998). The proposed mechanism is that less fat will be absorbed and therefore you will reduce your energy intake and consequently reduce your weight. The drawback with this lipase inhibitor is that it inhibits all types of lipases and produces steatorrhea due to strongly impaired fat digestion. It is therefore of outmost importance to develop a natural compound that retards fat digestion in a milder way without causing steatorrhea as side effect.
Cetilistat is another lipase inhibitor disclosed in WO00/40247, (Alizyme Therapeutics, 2000) with similar working mechanism, but with less side effects than orlistat (Kopelman P. et al., Int J Obes 2006; 31: 465-71).
Energy balance is a homeostatic system. Although malfunctions of this system can cause obesity, the relatively recent increase in the incidence of obesity is not thought to be the result of specific defects, but of a regulatory system unable to cope with the current context of cheap, high-energy foodstuffs, mechanized transport and non-manual labour. Commandeering elements of this regulatory system might provide the best opportunity for us to combat obesity (Murphy K. Bloom S., Nature 2006; 444: 854-859).
The “ileal brake” is a feedback mechanism activated by nutrients in the intestine, especially fat, with marked effects on satiety. Small amounts of fat are able to induce satiety and influence food intake (Welch I., et al. Gastroenterology 1985; 89: 1293-1297; Welch I., et al. Gut 1988; 29: 306-311; Greenberg D. and Smith, G. P., Psychosomatic medicine 1996; 58: 559-569; Maljaars P W J., et al, Int. J. Obesity 2008; 32: 1633-1639).
WO87/03198 refers to an enteric preparation in the form of a capsule or tablet containing a fat as the active substance that should be released in the intestine. This preparation utilizes the ileal brake mechanism.
WO99/02041 and WO2007/075142 disclose the use of emulsions based on fractionated palm oil and fractionated oat oil to bring fat far down in the intestine. This product (Olibra, DSM) is investigated in several studies (Burns A. A. et al., Int. J Obesity 2000; 24:1419-1425; Burns A. A. et al., Int. J Obesity 2001; 25:1487-1496; Burns A. A. et al., Eur J Clin Nutr 2002; 56:368-377; Logan C:M: et al., Eur J Clin Nutr 2006; 60:1081-1091; Diepvens K. et al., Int. J Obesity 2007; 31:942-949; Diepvens K. et al., Physiol Behav 2008; 95:114-117). A statistical significant effect is demonstrated in some cases. However, it is questionable if the effect can be considered as clinical.
Emulsions based on palm oil and proteins from partially denatured egg white, as disclosed in WO2006/053647, Unilever, and rapeseed oil or hydrogenated rapeseed oil together with milk protein and monoglycerides, as disclosed in WO2006/117069, Unilever, are also used to bring fat far down in intestine.
In WO2006/132586 (Albertsson) whole biological membranes or the hydrophobic peptides of biological membranes are used for the reduction of lipolytic activity and/or retard fat digestion, suppress appetite, body weight and/or lower blood lipids.
WO97/13500 refers to liposome formulations having incorporated therein an amount of a 5-β steroid effective to treat obesity, diabetes or hydrocortocoidism.
WO03/018529 refers to fatty-acid monoesters of an estrogen and a fatty acid for use in treatment of obesity and overweight. In a preferred pharmaceutical or cosmetic composition for intravenous injection the monoester is incorporated in a lipidic suspension prepared from liposomes.
JP2007204368 refers to a silk peptide as active ingredient useful in treatment of obesity, wherein the silk peptide is incorporated into a liposome.
Even if these products provide effective for treating obesity, there remains a need to provide improved methods for use in control and treatment of obesity and obesity-related diseases.
2.2.1 Proposed Mechanism and Initial Results in this Invention
Our process enable production of very small (smaller than 100 nm), uniform particles of unilamellar vesicle type. The mechanism for enhancement of satiety is assumed to be the following: These lipid particles, with extremely large surface area and very rich in galactolipids, interact with the lipases in the intestine and the reduced enzyme activity increases the concentration of lipids in the end of intestine. This activates the “ileal break mechanism” for satiety. We can control the enzyme activity and achieve sufficient satiety without any negative side effects. A particular advantage is that the lipids we use are components of a common food material, which eliminates any safety risks.
As a first step in proving this invention on satiety, a clinical evaluation under controlled conditions has been done. A morning meal containing a particle dispersion of oat lipid vesicles gave significant effects on GLP-1, which is a hormone in the blood and used as an indicator on satiety. This study is described in Example 7 below. An extended clinical study is now performed.
2.3. Encapsulation or Solubilization for Increasing Bioavailability of Valuable Nutrients in Foods and Feed
Within the field of formulation technology, various methods have been developed in order to solubilize encapsulate, and deliver biologically active materials. Our invention provides new application possibilities of nutrients or other important food components, such as antioxidants and aroma substances. These processes involve solubilization or encapsulation of the actual component in our particle carrier systems. In this way increased bioavailibility can be obtained
The most advanced applications in this field are found in drug delivery, which involves controlled administration of a pharmaceutical active material in order to achieve a therapeutic effect. An authority in this field Robert S, Lang stated that the current needs in this field are to reduce toxicity, to increase absorption and to improve release profile (C. M. Henry, Chem. & Engineering News 80 (2002) 39-47). Lipids are frequently applied, and the introduction of liposomes has proved to reduce toxicity in an application involving doxorubicin (the product Doxil is used in cancer therapy). A drug incorporated into a liposome is released by diffusion or released into the targeted cells by endocytosis. Some advanced recent studies are focused on drug targeting by attached ligands on the surface of the liposomes which bind to specific cell surface receptors. The general properties of lipid particles described in the present invention offer similar application possibilities as liposomes. They can be expected to be superior with regard to drug load and mechanical properties of the particles.
There are obviously many common features in gastrointestinal drug delivery and delivery of special food components. The present knowledge of delivery in food systems have recently been described in an excellent review by Sagalowicz and Leser (Delivery systems for liquid food products, Current Opinion in Colloid & Interface Science (2009), doi: 10.1016/j.cocis.2009.12.003). Sensitive molecules can be protected against degradation (such as oxidation during storage of the actual food product) and the most important aspect is that the bioavailability of important nutrients can be improved.
2.3.1 Conclusion Encapsulation and Solubilisation
The present invention provides new possibilities in this field. One application enables production of very small (smaller than 100 nm), uniform particles of self assembly type. Only limited mixing energy is required in the process. Also the preparation of particles of cubic lipid liquid-crystalline phases can be useful in this field. A wide range of molecules can be added into the particles, both hydrophilic and lipophilic molecules. A particular advantage is that the lipids we use are components of a common food material, which eliminates safety risks.
2.4 Method to Utilize the Full Emulsifying Capacity of Oils Containing Galactolipids in Emulsions
Already in the 1960s Stig Friberg demonstrated the important role of the lamellar liquid-crystalline phase in emulsion formation and stability. Now it is generally accepted that food emulsions stabilized by polar lipid must provide the lamellar liquid-crystalline phase during the emulsification process (cf. K. Larsson and Stig Friberg: Food Emulsion second ed. Marcel Dekker Inc., New York 1960).
The mechanism behind the emulsification is that the non-polar oil separates simultaneously as the lamellar liquid-crystalline phase is formed and the lamellar phase will then tend to coat the oil droplets spontaneously in order to reduce the surface energy of the dispersion. Such emulsions are kinetically stable at a solid content above about 40 vol %.
This also means that if the HII-phase is formed before the lamellar phase, there is a serious complication hindering the application of standard mechanical emulsification processes. The starting lipid material we use comprises galactolipids as a polar lipid component. Galactolipids may form HII-particles. Using traditional emulsifying methods on oils containing galactolipids may fail or become difficult to reproduce because formation of HII-particles. Large amounts of energy are required to emulsify oil in water. It is difficult to get the particles small enough, to get a sufficient narrow particle size distribution and the variation between batches are very high.
By using the methods presented in this invention we can circumvent the ranges where HII particles are formed and the energy consumption is reduced, the particle size is reduced, the particle size distribution is narrower and the variation between batches is reduced.
2.5 Removal of Ethanol from Oils Containing Galactolipids without Forming HII-Particles
When ethanol is removed from oil fractions containing galactolipids and ethanol there is a risk that HII-particles are formed. Once formed it may take years before the HII-particles are dissolved. This means that oils containing HII-particles lose their emulsifying properties.
Therefore there is a great need to find methods to remove ethanol from oils containing galactolipids without forming HII-particles.
By using the methods presented in this invention we can; circumvent the ranges where HII particles are formed during removal of ethanol from oils containing galactolipids; reduce energy consumption, the particle size, the particle size distribution and reduce the variation in product quality between batches in downstream processes.